• • Three monoclonal IgA-knockout porcine fetal fibroblast lines were generated with precise deletions of 1044 bp (heterozygous), 1043 bp (homozygous), and 1039 bp (homozygous) in the CH1-CH3 region, demonstrating high editing efficiency and genotype diversity without transgene integration.
• • SCNT using pooled donor cells yielded 400 fused cells from 500 enucleated oocytes (80% fusion rate), and transfer of 300 embryos into a surrogate sow resulted in a 28-day pregnancy confirmation and birth of six live F0 piglets after 143 days of gestation, achieving a 2% birth rate per transferred embryo.
• • Genotyping of F0 piglets confirmed two heterozygotes (4010#, 4015#) and four homozygous knockouts, with exactly two piglets per genotype (1044, 1043, 1039 bp deletions), validating the deliberate mixing of donor cells to obtain multiple genotypes in a single pregnancy.
• • The IRE-DSRNP method bypasses plasmid DNA, eliminating resistance gene integration and reducing off-target cleavage risks, while shortening the cell line acquisition period from 3–4 weeks to a rapid, transgene-free workflow, directly addressing biosafety and timeline bottlenecks in transgenic pig production.